Research
Focused on an alternative method to breach skin and cell membranes: the use of pressure waves to transiently open channels through those barriers, so that molecules can diffuse rapidly before they reversible close (within few minutes). Photoacoustic pressure waves may approach the ideal method for transdermal drug delivery because they are painless, do not lead to allergies or contaminations, and the skin rapidly recovers after the drug is administered. They may be the only direct method known that permeate the nuclear membrane and offer valuable alternative for in-vivo cell membrane controlled permeation. The efficacy of the acoustic wave depends on its form, that is, intensity, rise-time, duration and peak pressure. The efficient delivery of a relatively large molecule requires that a very high pressure is applied for a very short period of time onto the biological material. When the photoacoustic wave generated by an appropriate material reaches the barrier, it produces an "earthquake" that transiently disturbs its structure and increases its permeation to large molecules. The best method to generate such an acoustic wave is by the absorption of very short laser pulses by an appropriate material. The radiative energy transiently heats the material, causes its thermal expansion and, in a confined space, efficiently originates a pressure wave. We develop new tailor made materials based on designed geometries and including dyes or carbon with adequate morphology.
Tailoring Molecules and Materials for Sustainable Applications
One of the major challenges in the field of Biophysical Chemistry is the study of protein folding mechanisms, i.e., how an unstructured polypeptide chain can rapidly adopt a unique, densely packed, three dimensional structure. Erroneous folding is the molecular basis for a wide range of human disorders, such as neurodegenerative diseases including Alzheimer's and Parkinson's disease. The earliest conformational events related to folding, such as the formation of the isolated helical segments, reverse turns and β hairpins, occur within microseconds or less. Our aim is to make the early stages of folding experimentally accessible, so we need to initiate those processes in shorter timescale than the fastest structural event of interest. We use ultrafast pH-jump technique to induce peptide and protein unfolding. The conformational changes during unfolding are monitored by time-resolved photoacoustics calorimetry (TR-PAC), enabling the determination of kinetic constants, enthalpy and volume changes accompanying the unfolding process. PAC is used in complement with other fast detection techniques (fluorescence and transient absorption spectroscopy) and structural techniques (NMR, CD, Ultrasound Absorption). We presently focus our study on model peptides with well defined secondary structure and proteins susceptible of pH unfolding.
Amyloids are defined as a class of supramolecular assemblies of misfolded proteins or peptides into β-sheet fibrils, and their deposition in tissues and organs is associated with several medical disorders, including Parkinson’s disease and Alzheimer’s disease. Searching for molecular agents that have a therapeutic impact on amyloid aggregates, several different classes of small molecules have been investigated by preventing or retarding their formation, but this is still an unfulfilled goal. There is a great demand for developing fast and reliable methods for in vitro fast screening of new drugs that may suppress or reverse amyloidogenesis. It has been recently unravelled that amyloidogenic protein aggregation induces a new emission band in the deep blue region (∼450 nm) upon excitation at the edge of the long UV wavelength range (∼350 nm). It is recognized that a progressive increase in the fluorescence emission in the visible region is concomitant with the growth of amyloid protein aggregates. So far, peptides and proteins as α-synuclein, β-amyloid(1-40),(1-42), tau, lysozyme or insulin shown to exhibit auto-fluorescence accompanied by amyloid fibril assembly. We want to contribute to the understanding of the nature of the aggregates formed during amyloidal fibrillation. Correlate the nature of amyloidal fibrils with the observed intrinsic auto-fluorescence in conditions of natural fibrillation and presence of aggregation inhibitors; investigate aggregation accelerators (Cu or Zn); and develop fluorescence micro-plate reader in vitro screening method for amyloid inhibitors.
Human serum albumin (HSA) is a monomeric multidomain biomacromolecule considered the most abundant globular protein in human plasma with a normal concentration in the 30-50 g/L range. Among the biological importance of HSA highlights the capacity to carrier different endogenous and exogenous compounds, e.g., fatty acids, bilirubin, vitamins, drugs, hormones, steroids, and porphyrins, impacting the pharmacokinetic profile of clinical approved and potential drugs. The interaction between HSA and molecules/drugs is scrutinized using multiple spectroscopic techniques, including UV-visible absorption, circular dichroism, steady-state, and time-resolved fluorescence under physiological conditions. Calorimetric data from isothermal titration calorimetry is also used to determine the thermodynamic parameters of interactions. All the spectroscopic results, including experimental drug-displacement assays, are correlated with molecular docking calculations and molecular dynamic simulation to offer a molecular-level explanation of the binding capacity.
The basis of the Photoacoustic (PA) effect is the non-radiative release of part of the light absorbed by the molecules in a given material, resulting in local heating and thus the generation of a pressure (or acoustic) wave propagating away from the heat source. Since the discovery of the PA effect, in 1880 by A. G. Bell, techniques related to its application have evolved to become very efficient methods for thermal and optical characterization of light absorbing materials. The comprehensive description of the PA effect in solids and liquids, the invention of intense laser sources, and the development of highly sensitive acoustic detectors contributed to those advances.
In our Laboratory we have an accumulated experience of 30 years on time resolved Photoacoustic Calorimetry (TR-PAC). This technique was initially devised for studies in solution, but was recently extended to solid:liquid interfaces. New setups and methodologies have been developed for that purpose, and proved useful for the characterization of photoelectrochemical cells.
The intensity of the PA signal depends on the sample absorption coefficients at the incident light wavelength, on the efficiency of conversion of the incident light into heat, and on how the heat diffuses through the sample. Spectroscopic information can be obtained from the first dependency, and the absorption spectra of opaque solids acquired. The information about the non-radiative decay channels is the basis of TR-PAC, allowing the determination of enthalpic, kinetic and volume changes in photoinduced processes. Over the years we applied this technique to electron and proton transfer reactions, to the photophysical study of triplet states, to energy transfer reactions with singlet oxygen formation and many others. We are using TR-PAC to follow conformational changes in peptides and proteins. Reconstruction of the heat defunded by an irradiated source is the basis of the imaging technique denominated Photoacoustic Tomography. This technique installed in our laboratories has great potential for applications in Biomedicine as it combines the advantages of acoustic resolution detection with a light absorptive contrast agent.